The Journal of Neuroscience, January 19, 2011 • 31(3):1093–1105 • 1093
Cellular/Molecular
Frequency-Dependent Modes of Synaptic Vesicle
Endocytosis and Exocytosis at Adult Mouse
Neuromuscular Junctions
Yuka Maeno-Hikichi, Luis Polo-Parada, Ksenia V. Kastanenka, and Lynn T. Landmesser
Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4975
During locomotion, adult rodent lumbar motoneurons fire in high-frequency (80 –100 Hz) 1–2 s bursts every several seconds, releasing
between 10,000 and 20,000 vesicles per burst. The estimated total vesicle pool size indicates that all vesicles would be used within 30 s;
thus, a mechanism for rapid endocytosis and vesicle recycling is necessary to maintain effective transmission and motor behavior.
However, whether such rapid recycling exists at mouse neuromuscular junctions (NMJs) or how it is regulated has been unclear. Here, we
show that much less FM1-43 dye is lost per stimulus with 100 Hz stimulation than with 10 Hz stimulation even when the same number of
vesicles undergo exocytosis. Electrophysiological data using folimycin show this lesser amount of dye loss is caused in part by the rapid
reuse of vesicles. We showed previously that a myosin light chain kinase (MLCK)–myosin II pathway was required for effective transmission at 100 Hz. Here, we confirm the activation of MLCK, based on increased nerve terminal phospho-MLC immunostaining, with 100
Hz but not with 10 Hz stimulation. We further demonstrate that activation of MLCK, by increased extracellular Ca 2⫹, by PKC (protein
kinase C) activation, or by a MLCK agonist peptide, reduces the amount of dye lost even with 10 Hz stimulation. MLCK activation at 10 Hz
also resulted in more vesicles being rapidly reused. Thus, MLCK activation by 100 Hz stimulation switches the mechanism of vesicle
cycling to a rapid-reuse mode and is required to sustain effective transmission in adult mouse NMJs.
Introduction
The adult vertebrate neuromuscular junction (NMJ) is a highly
reliable synapse that must exocytose a large number of vesicles
with each stimulus to ensure effective excitation of the postsynaptic muscle fiber. During normal locomotor movements, most
motoneurons are activated for only brief periods (1–2 s) with
similar duration intervening periods of rest. In addition, fast rat
motoneurons fire in vivo at frequencies of between 80 and 100 Hz
with even higher frequencies at the beginning of bursts (Gorassini
et al., 2000). Since the quantal content of the mouse fast semitendinosus junctions used in the present study is ⬃100 (Polo-Parada
et al., 2001), a 1 s 100 Hz burst, which mimics in vivo locomotor
activity, would trigger the release of ⬃10,000 vesicles. To maintain effective synaptic transmission, the NMJ must be able to
resupply an equivalent number of vesicles by subsequent endocytosis or by recruitment from the reserve pool. Since the size of
Received June 2, 2010; revised Sept. 27, 2010; accepted Nov. 9, 2010.
This work was supported by National Institutes of Health–National Institute of Neurological Disorders and Stroke
Grants NS19640 and NS23678 (L.T.L.) and by American Heart Association Grant 0530140 (L.P.-P.). We thank Katherine Lobur and Smaranda Ene for their excellent technical assistance and Katsusuke Hata and Sheng Wang for their
input throughout this work. We also thank Guillermo Pilar, David Friel, and Corey Smith for their comments on this
manuscript. The mAb 35 developed by J. Lindstrom was obtained from the Developmental Studies Hybridoma Bank
developed under the auspices of the National Institute of Child Health and Human Development and maintained by
Department of Biology, University of Iowa (Iowa City, IA).
Correspondence should be addressed to Lynn T. Landmesser, Department of Neurosciences, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4975. E-mail: [email protected].
L. Polo-Parada’s present address: Department of Medical Pharmacology and Physiology, Dalton Cardiovascular
Research Center, University of Missouri, 134 Research Park Drive, Columbia, MO 65211.
DOI:10.1523/JNEUROSCI.2800-10.2011
Copyright © 2011 the authors 0270-6474/11/311093-13$15.00/0
the total pool of synaptic vesicles (sum of reserve pool, recycling pool, and readily releasable pool) at rodent NMJs has
been estimated to be between 174,000 and 366,000 (Elmqvist
and Quastel, 1965; Schofield and Marshall, 1980; Reid et al.,
1999), in the absence of a mechanism for replenishment, at
release rates needed to sustain transmission during in vivo
locomotion, all vesicles would be used within ⬃1 min. Therefore, a mechanism for very rapid vesicle recycling must exist to
maintain effective transmission when terminals are activated
with high-frequency repetitive stimulation. We previously
identified an intracellular signaling pathway involving myosin
light chain kinase (MLCK) and myosin II as being required for
effective transmission at adult mouse NMJs at high (100 Hz
and above) but not at low (10 Hz) stimulus frequencies (PoloParada et al., 2001, 2004, 2005). When this pathway is blocked,
the junctions are activated normally at 10 Hz, but they exhibit
cyclical periods of transmission failures at 100 Hz (PoloParada et al., 2001, 2004, 2005). Thus, it appears that the
junctions are normally able to switch from a mode of transmission that does not require the MLCK pathway to one that
does, depending on the frequency of stimulation. To further
explore the distinctive mechanisms of vesicle cycling used at
each frequency and the signal(s) that might trigger the switch
in cycling mechanisms, we studied adult mouse NMJs by electrophysiology and by using styryl dyes (Betz et al., 1992, 1996;
Cousin and Robinson, 1999; Ryan, 2001; Gaffield and Betz,
2006) to assess the synaptic vesicle exocytosis and endocytosis
under different conditions.
Maeno-Hikichi et al. • Synaptic Vesicle Endocytosis/Exocytosis at Mouse NMJs
1094 • J. Neurosci., January 19, 2011 • 31(3):1093–1105
Materials and Methods
Tissue preparation. All experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of
Case Western Reserve University, an accredited oversight body (Federal
Animal Welfare Assurance No. A3145-01), in accordance with National
Institutes of Health guidelines. Semitendinosus muscles with attached
nerves were dissected from 1- to 2-month-old adult C57BL/6 mice (inbred; originally from The Jackson Laboratory). Animals were killed by
carbon dioxide and decapitated. Muscles were immediately removed and
immersed in ice-cold Tyrode’s solution [125 mM NaCl, 5 mM KCl, 24 mM
NaHCO3, 1 mM MgCl2, 10 mM glucose (Sigma-Aldrich), 2 mM CaCl2]
that was oxygenated by 95% O2–5% CO2 (all chemicals were from
Thermo Fisher Scientific, unless otherwise noted). Muscles were cleaned
by removing excess fat and connective tissue and then kept in oxygenated
Tyrode’s until needed.
Peptide introduction into adult NMJs. Peptides were introduced into
the semitendinosus nerve–muscle preparation by using the peptide carrier Chariot (Active Motif) according to the manufacturer’s instructions.
Briefly, a Chariot stock solution (2 mg/ml) was prepared, aliquoted, and
kept at ⫺70°C. The frozen stock solution was thawed and sonicated 3
min in a water bath and mixed with the peptide solution at a peptide/
chariot mole ratio of 1:4 and incubated at room temperature for 1 h. The
final peptide concentration in the bath was adjusted to 3.5 ␮M. The sequences of peptides used are KKDRMKKYMA (synthesized by the Analysis Department, Wolfson Institute for Biomedical Research, London,
UK) for the MLCK agonist peptide and EAVSLKPT (Calbiochem) for the
protein kinase C␧ (PKC␧) translocation inhibitor peptide.
Electrophysiological recordings. Electrophysiological recordings were
conducted using standard methods from acutely isolated semitendinosus
muscles as described previously (Polo-Parada et al., 2005). The muscle–
nerve preparation was gently extended and pinned flat in a Sylgard (Dow
Corning)-coated recording chamber, perfused with normal Tyrode’s solution, and gassed with 95% O2–5% CO2. To prevent muscle contraction, 1–2 ␮M ␮-conotoxin GIIIB (muscle-specific voltage-gated sodium
channel blocker) (Alomone Labs) was added to the perfusion buffer.
Sharp glass electrodes were pulled (10 –20 M⍀), filled with 3 M KCl, and
single muscle fibers were impaled near the motor endplate. Potentials
were recorded via an intracellular amplifier (World Precision Instruments) using Axoscope software (40 kHz sampling rate; Applied Biosystems/MDS Analytical Technologies). The nerves were stimulated via a
suction electrode pulled from polyethylene tubing (PE-190; BD Biosciences) and a SQ38 stimulator and PSIU6B stimulus isolation unit
(Grass Technologies). The MLCK agonist peptide or the PKC␧ translocation inhibitor peptide was introduced into the bath 1 h before and was
kept in the bath during the recordings. Folimycin (EMD Chemicals; final
concentration of 160 nM) was added to the bath 1 min before the recordings and kept in the bath throughout the recordings. Recordings were
made within 30 min after applying the folimycin to prevent the drug
from affecting vesicles that had not undergone exocytosis. Eighty micromolar dynasore (Sigma-Aldrich) was used to block dynamin 1 and 2
activity, and was applied 30 min before and throughout the recordings.
Immunohistochemistry. Semitendinosus muscles were dissected and
quickly fixed in 3.7% formaldehyde (Sigma-Aldrich) in PBS (140 mM
NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) at room temperature for 30 min, washed with PBS, and cryoprotected in 30% sucrose in
PBS overnight at 4°C. Fifty micrometer longitudinal frozen sections or 12
␮m cross-sections were cut, mounted on gelatin-coated slides, air-dried,
and stored at ⫺70°C. Sections were permeabilized and blocked with 2%
bovine serum albumin (Sigma-Aldrich), 10% goat serum, and 0.5% Triton X-100 in PBS at room temperature for 1 h, then incubated with
primary antibodies at either room temperature for 2 h or at 4°C overnight, washed, and then incubated at room temperature for 2 h with
fluorochrome-conjugated secondary antibodies (Zymed) and with
fluorochrome-conjugated ␣-bungarotoxin (Invitrogen) to visualize the
postsynaptic acetylcholine receptors. The primary antibodies used were
phospho-MLC2 (T18/S19) (Cell Signaling Technology) and PKC␧
(Santa Cruz). For the phorbol 12-myristate 13-acetate (PMA) treatment,
100 nM PMA (Sigma-Aldrich) was added to the bath for 1 min before
rapid fixation of the muscle. Stimulated samples were prepared either by
stimulating the nerve–muscle preparation at 10 Hz for 10 min or at 100
Hz for 1 min and replacing the Tyrode’s solution with 3.7% formaldehyde in PBS 30 s before the end of stimulation, followed by an additional
30 min fixation. For peptide treatment, muscles were preincubated with
the peptide–Chariot complex for 1 h and then stimulated at either 10 or
100 Hz. To prevent dephosphorylation, a phosphatase inhibitor tablet,
PhosSTOP (Roche), was added to the fixing solution.
Quantification of immunohistochemistry. After immunostaining, pictures were acquired with a BX51WI Olympus microscope or a Nikon
Microphoto-FX (Nikon Instruments) equipped with Retiga EXi CCD
camera (QImaging) using the same exposure time between samples.
Quantification of pixel intensity was performed with the MetaMorph
Imaging System (Molecular Devices) on defined regions of interest using
the same parameters between samples. Background nonspecific muscle
staining/autofluorescence was subtracted from each picture by using the
same sized region of interest and an area adjacent to the endplate on the
same muscle.
FM dye optical imaging. The nerve–muscle preparation was pinned flat
in a Sylgard-coated recording chamber, perfused with normal Tyrode’s
solution, and gassed with 95% O2–5% CO2. Endplates were labeled with
the nicotinic acetylcholine receptor antibody mAb 35 (Developmental
Studies Hybridoma Bank) conjugated with Alexa 546 fluorochrome (Invitrogen) for 1 h together with ␮-conotoxin GIIIB to block contractility.
The recycling pool of synaptic vesicles was loaded with either FM1-43 or
FM2-10 (both from Invitrogen) by stimulating the nerve at either 10 Hz
for 30 min or 100 Hz for 3 min in normal Tyrode’s solution with 8 ␮M
FM1-43 or 100 ␮M FM2-10. Some short-term stimulation protocols used
either 10 Hz for 10 min or 100 Hz for 1 min. The muscles were incubated
in the FM dye for 5 min after the stimulus to allow any slow endocytosis
to be completed. The sample was then washed with low-calcium, highmagnesium Tyrode’s three times, incubated with ADVASEP-7 (Biotium)
for 5 min, washed again, and perfused at least 20 min with low-calcium
high-magnesium Tyrode’s. To unload FM dye, the bath solution was
replaced with normal calcium Tyrode’s and perfused for 5–10 min, and
then stimulated at either 10 or 100 Hz. The MLCK agonist peptide was
added after complete loading and washing as described above during a
1 h incubation. The buffer was replaced with normal Tyrode’s before dye
destaining. To see the effect of PMA on FM destaining, 100 nM PMA was
added to the bath before the destaining stimulation and when the buffer
was replaced with normal Tyrode’s with drug. PMA was kept in the
buffer during the destaining stimulation. To see effects of different drugs
on loading at 100 Hz, nifedipine (final concentration of 5 ␮M) or BFA (10
␮g/ml) were added into the bath for 1 h preincubation before the loading. Botulinum neurotoxin A (BoNTXA) was activated with 5 mM DTT
for 1 h, and then added to the bath for a final concentration of 5 nM 3 h
before the start of loading.
Data presentation and analysis. All data are presented as mean ⫾ SE.
The statistical significance was calculated by one-way ANOVA unless
noted otherwise and is presented as **p ⬍ 0.01 or *p ⬍ 0.05. In many
experiments, n refers to the number of separate experiments and is presented as Nexp (N ⫽ number of experiments). In other cases, in which
multiple endplates were assessed in one or more muscles, n is also presented as nend (n ⫽ number of endplates).
Results
Characteristics of endocytosis at adult mouse neuromuscular
junctions with 10 Hz stimulation
Sustained effective transmission relies on a balance between exocytosis and endocytosis and the speed of synaptic vesicle recycling. To examine these different components, we monitored the
FM dye uptake and destaining at adult mouse NMJs. Since FM
dye destaining will, in part, be based on the behavior of the pool
of vesicles previously loaded with dye, we first examined dye
uptake with 10 Hz stimulation. To mimic the physiological condition, we used a series of 1 s trains at 10 Hz with intervening 1 s
rest periods (1 s trains at 0.5 Hz) (Fig. 1 A). Although both exocytosis and endocytosis occur during the stimulation period, en-
Maeno-Hikichi et al. • Synaptic Vesicle Endocytosis/Exocytosis at Mouse NMJs
Figure 1. FM1-43 uptake at the adult mouse neuromuscular junction. A, Schematic diagram
of the stimulation and FM dye loading protocol. B, FM1-43 uptake with a 10 Hz, 30 min stimulation. The error bars indicate SE throughout the study (nend ⫽ 17; Nexp ⫽ 17). C, A bar graph of
final pixel intensity after completion of uptake when dye was present only during the stimulus
or for an additional 5 min after the end of the stimulus. Endpoint pixel intensity was measured
after washing out nonspecific binding of FMs on the surface membrane and thus reflects only
internalized FMs. Mean values at 10 Hz were 8208 ⫾ 759 (nend ⫽ 30; Nexp ⫽ 2) and 11,044 ⫾
1062 (nend ⫽ 63; Nexp ⫽ 15) for loading only during the stimulus or with 5 min poststimulus
dye incubation, respectively. Statistical significance was indicated throughout the study as
follows: **p ⬍ 0.01 or *p ⬍ 0.05.
docytosis can continue after the end of stimulation (de Lange et
al., 2003; Royle and Lagnado, 2003), especially in cases in which
the endocytic process is not rapid enough to retrieve all the vesicle
membrane added to the terminal surface membrane during the
stimulus period. Therefore, we compared dye uptake when
FM1-43 was in the bath only during the period of the 9000 stimuli
(Fig. 1 A, B) and when the dye remained in the bath for 5 min after
stimulus termination (Fig. 1 A, C). In experiments in which dye
uptake was measured while FM dye was still in the bath (Figs. 1 B,
4 A), the amount of uptake will include both dye taken into vesicles and cell surface labeling. However, for simplicity, we refer to
this simply as “uptake.” To characterize the frequency dependence, we plotted dye uptake or loss per stimulus event and not as
a function of time throughout this work, and related the change
in dye uptake or loss to the number of vesicles exocytosed, based
on summed quantal content.
When NMJs were loaded with FM1-43 by 10 Hz stimulation,
dye uptake occurred in an asymptotic manner and reached a
plateau after ⬃15 min (4000 – 6000 stimuli) (Fig. 1 B). At 10 Hz
stimulation, a slight increase of dye uptake was also observed after
stimulus cessation (Fig. 1C). The mean intensity values after wash,
which will remove surface labeling, were 8208 ⫾ 759 (nend ⫽ 30;
Nexp ⫽ 2) at the end of 30 min of stimulation, with an additional
increase to 11,044 ⫾ 1062 (nend ⫽ 63; Nexp ⫽ 15) after a 5 min
poststimulus loading period. Although the mean intensity increased by 2836 ⫾ 1821 (26% of the total dye) after stimulus
cessation, the total mean intensity at the end of loading period did
not differ significantly from that at the end of stimulation ( p ⬎
0.05). Thus, most of the endocytosis triggered by 10 Hz stimulation is completed during the stimulation period.
J. Neurosci., January 19, 2011 • 31(3):1093–1105 • 1095
The amount of FM1-43 dye loss differs at 10 and 100 Hz even
for the same number of vesicles undergoing exocytosis
To examine the process of exocytosis at different frequencies of
stimulation, we characterized the amount of dye loss from vesicle
pools that were loaded with 10 Hz stimulation. To maximize dye
loading into all releasable vesicle pools, unless noted otherwise,
our standard loading protocol was 10 Hz stimulation for 30 min
(1 s 10 Hz trains at 0.5 Hz; 9000 stimuli) with dye remaining in
the bath for an additional 5 min (Fig. 1 A). After a wash in lowCa 2⫹ Tyrode’s solution with ADVASEP-7 to remove any cell
surface label, the amount of dye loss from the loaded vesicles was
assessed by stimulating the endplates with 1 s trains of either 10 or
100 Hz, with 1 s alternating periods of rest, a pattern that approximates the way in which these junctions would be activated in vivo
(Gorassini et al., 2000) (Fig. 1 A).
At 10 Hz, dye loss was rapid with ⬃50% of the dye being
released by the 3000th stimulus (Fig. 2 A). However, with 100 Hz,
much less dye was lost with only ⬃20% of the dye being lost by
the 3000th stimulus (i.e., during the first minute of stimulation).
One possible explanation for this observation is that fewer vesicles may have undergone exocytosis during the high-frequency
stimulation because of depression, and it was thus necessary to
correct for this. However, with repetitive trains of high-frequency
stimulation, a greater proportion of vesicles are released asynchronously both during the stimulus and during the resting period, and substantial asynchronous release usually continued for
some time after each train as well (Fig. 2 B, top trace, arrowhead)
(David and Barrett, 2003). We therefore could not simply correct
for depression by cumulatively summing quantal contents based
on endplate potential (EPP) amplitudes.
To take into account both depression and asynchronous release and thus arrive at the best estimate of the total number of
vesicles exocytosed during the stimulation at 10 Hz versus 100
Hz, electrical recordings were made from endplates stimulated
with the same protocols used to unload FM1-43 dye (Fig. 2 B). To
estimate the total number of quanta/vesicles that had undergone
exocytosis at each stimulus frequency by the 3000th stimulus, a
time point at which a significant difference in the amount of dye
lost at the two frequencies was apparent, we measured the area
under the voltage trace that was above the baseline resting
potential for each train (in millivolts 䡠 millisecond), including the
enhanced asynchronous release at the ends of the high-frequency
trains and divided this by the mean area (in millivolts 䡠 millisecond)
of the individual miniature endplate potentials (mepps) recorded
before the stimulation. This provided us with an estimate of the
total number of quanta contributing to the electrical response
that had been released at different points during the 10 and 100
Hz stimulation. However, at 100 Hz, there was also some increase
in baseline. This was partly attributable to EPPs occurring before
the previous one had decayed to baseline as well as asynchronous
release. Since it was difficult to distinguish how much the increase
in baseline was attributable to the summation of voltage from
successive EPPs versus asynchronous release, we corrected the
area under the curve during the stimulation by the amount of the
increased baseline, which reduced by ⬃30% the original calculation of the summed quantal content. However, this is clearly an
overcorrection as much of the increase in baseline, especially toward the end of train was attributable to asynchronous release.
The corrected calculated quanta were summed and plotted
against stimulus number at every 100th stimulus (Fig. 2C). The
data show that, by the 3000th stimulus, fewer vesicles had been
released with the 100 Hz stimulation; however, until the 100 –
200th stimulus, the number of quanta/vesicles that were released
1096 • J. Neurosci., January 19, 2011 • 31(3):1093–1105
Maeno-Hikichi et al. • Synaptic Vesicle Endocytosis/Exocytosis at Mouse NMJs
did not differ appreciably between the 10
and 100 Hz stimulation. To determine
whether the lesser amount of dye loss observed with 100 Hz stimulation (Fig. 2A)
could be accounted for by fewer vesicles undergoing exocytosis, we performed a
FM1-43 destaining experiment with greater
temporal resolution for the first 500 stimuli.
Figure 2D shows that during the first 100 –
200 stimuli, dye loss was greater with 10 Hz
compared with 100 Hz stimulation, similar
to the data shown in Figure 2A. We then
plotted the average normalized FM intensity
at different time points against the summed
quantal content at each point (Fig. 2 E).
With the 10 Hz stimulation, the summed
quantal content and FM dye loss had a linear
relationship. However, with 100 Hz stimulation, the relationship indicated that there
was less dye loss than would be expected
from the amount of transmitter released. In
fact, virtually no dye loss was observed even
after 10,000 quanta/vesicles had undergone
exocytosis.
FM2-10 dye unloading is similar to
that of FM1-43 both with 10 and 100
Hz stimulation
What might account for the different rates
of dye loss at the different stimulation frequencies? Several possibilities were considered. First, the actual mechanism of vesicle
fusion might differ during 10 Hz versus 100
Hz stimulation, resulting in different
amounts of dye loss, even though the same
amount of transmitter was being released.
Second, different pools of vesicles might undergo exocytosis with the different frequencies of stimulation. For example, the 100 Hz
stimulus might mobilize vesicles from a Figure 2. FM1-43 destaining with 10 and 100 Hz stimulation. A, FM1-43 destaining at 10 Hz (white circles; nend ⫽ 7; Nexp ⫽ 7)
pool that was not loaded by the standard 10 and 100 Hz (black circles; nend ⫽ 6; Nexp ⫽ 6). In both cases, loading was with 10 Hz stimulation for 30 min with 5 min poststimulus
Hz loading protocol, and exocytosis of these dye incubation. Destaining was performed with 1 s trains separated by 1 s rest periods the same as the loading protocol. B, EPP
vesicles would maintain transmission but traces with 10 or 100 Hz stimulation showing the beginning and the end of the first train (for both the 10 and 100 Hz stimulation,
not result in dye loss. Third, a greater pro- the endplates received a total of 3000 stimuli). In the top trace, the arrow indicates asynchronous release during an EPP, and the
portion of vesicles that had already released arrowhead indicates extensive asynchronous release at the end of the train. The arrowheads in the bottom trace indicate mepps,
but these were included in our measurements of asynchronous release if they occurred during the train. C, A graph of summed
their dye might be rapidly recycled, filled quantal content plotted against the number of stimuli and calculated as described in the text. White circles, 10 Hz (n ⫽ 4; N
end
exp
with transmitter, and exocytosed again dur- ⫽ 4); black circles, 100 Hz (n ⫽ 5; N ⫽ 5). Depression was noticeable after 200 stimuli with 100 Hz stimulation.
D, FM1-43
end
exp
ing the 100 Hz stimulation. Such vesicles destaining at 10 Hz (white circles; nend ⫽ 5; Nexp ⫽ 5) and 100 Hz (black circles; nend ⫽ 12; Nexp ⫽ 12) during the first 500 stimuli.
could maintain transmission but would not E, A graph of normalized loss of FM dye intensity plotted against the summed quantal content, calculated from the average values
contribute to dye loss.
from A and C. White circles, 10 Hz; black circles, 100 Hz. The lines are regression curves calculated from the data and fitted to either
Several modes of vesicle fusion and re- a linear (10 Hz) or a polynomial (100 Hz) curve using SigmaPlot software.
cycling have been described (Rizzoli and
apse, and the stimulus conditions (Neher and Marty, 1982;
Betz, 2005; Harata et al., 2006b; He and Wu, 2007; Rizzoli and
Klingauf et al., 1998; Alés et al., 1999; Stevens and Williams,
Jahn, 2007; Zhu et al., 2009) as indicated in Figure 3A. In one, the
2000; Klyachko and Jackson, 2002; Wang et al., 2003; Fulop et
vesicle fully collapses into the terminal membrane after fusion,
al., 2005) and both can coexist in a given nerve terminal (Koereleasing all of its contents, including transmitter and FM dye.
nig and Ikeda, 1996; Richards et al., 2000, 2003; Gandhi and
This has been referred to as classical full collapse fusion. In the
Stevens, 2003) (Fig. 3A).
second, termed “kiss-and-run” or “transient fusion,” a small
Since there is considerable evidence to support full collapse
transient fusion pore connects the vesicle interior to the outside
solution, resulting in the release of transmitter, but the pore
exocytosis at many synapses, at least under the stimulation procloses before all of the FM dye can departition from the lipid of
tocols we used, we made the initial assumption in our studies,
the vesicle membrane. The importance of these two modes of
that full collapse exocytosis was used at both the 10 and 100 Hz
exocytosis varies depending on the species, the type of synstimulation frequencies. Several possibilities then exist for why
Maeno-Hikichi et al. • Synaptic Vesicle Endocytosis/Exocytosis at Mouse NMJs
Figure 3. Synaptic vesicle exocytosis and endocytosis at the NMJ. A, Schematic model of
possible modes of vesicle exocytosis and endocytosis at the adult mouse neuromuscular
junction. Note that the existence of compound vesicles and release by compound exocytosis has not yet been demonstrated at the NMJ. B, FM2-10 destaining at 10 Hz (white
circles; nend ⫽ 5; Nexp ⫽ 5) and 100 Hz (black circles; nend ⫽ 5; Nexp ⫽ 5).
FM1-43 dye destaining was much less at 100 Hz than was expected from the number of vesicles undergoing fusion. First,
there might be insufficient time for the dye to departition from
the cell surface membrane after full collapse exocytosis because
of the high rate of vesicle membrane addition with 100 Hz stimulation. To determine whether slow diffusion of FM1-43 from
the membrane could explain the smaller amount of dye loss, we
performed similar experiments using FM2-10, which diffuses
four times faster than FM1-43. The measured dissociation times
have been reported as 2.7 s for FM1-43 and 0.7 s for FM2-10
(Ryan et al., 1996). As shown in Figure 3B, the amount of dye loss
for FM2-10 was also less at 100 Hz compared with at 10 Hz, not
differing appreciably for the dye loss of FM1-43 (Fig. 2 A). We
also performed a similar high-frequency stimulation protocol,
but increased the intervening periods of rest 10 times, thus allowing more time for FM1-43 to diffuse from the surface membrane
before making the fluorescence measurements. However, this
also did not alter the dye loss with 100 Hz stimulation (data not
shown). These observations suggest that the less amount of dye
lost at 100 Hz stimulation was not attributable to the slow diffusion of FM1-43 from the cell surface membrane but rather that
different mechanisms of exocytosis or reuse were used at the two
frequencies.
J. Neurosci., January 19, 2011 • 31(3):1093–1105 • 1097
Characteristics of endocytosis at adult mouse neuromuscular
junctions with 100 Hz stimulation
We next addressed the possibility that a different pool of vesicles,
which was not loaded with 10 Hz stimulation, underwent exocytosis during the 100 Hz stimulation, thus contributing to the slow
dye loss seen at this frequency. To load such vesicle pools with
FM1-43, we used a 100 Hz stimulus with 5 min poststimulus
uptake (Fig. 1 A), since such intense stimulation has been shown
to mobilize vesicles that do not undergo exocytosis with less intense, lower frequency stimulation (Rizzoli and Betz, 2005). First,
we examined the vesicles loaded with 100 Hz stimulation to see
whether they behaved similarly to those loaded with 10 Hz. In
contrast to 10 Hz stimulation (Fig. 1 B), with the 100 Hz stimulation, dye uptake occurred more linearly during the stimulation
period, with a greater variability among the endplates, especially
at early stages of stimulation (Fig. 4 A). This suggests that the
degree of dye uptake varied more between endplates at 100 Hz
compared with 10 Hz stimulation early in the stimulation period.
Also, there was considerable dye uptake after cessation of the 100
Hz stimulus (Fig. 4 B). Such uptake likely corresponds to a bulk
form of endocytosis described by others (Heuser and Reese, 1973;
Miller and Heuser, 1984; Koenig and Ikeda, 1996; Richards et al.,
2003; Jockusch et al., 2005; Rizzoli and Betz, 2005) (Fig. 3A). The
mean pixel intensity values after wash were 5151 ⫾ 343 (nend ⫽
91; Nexp ⫽ 3) versus 17,016 ⫾ 1595 (nend ⫽ 68; Nexp ⫽ 7) for dye
uptake without and with 5 min poststimulus dye application,
respectively (Fig. 4 B), and these were the statistically significant
( p ⬍ 0.01). With 100 Hz stimulation, the mean pixel intensity
increased by 11,865 ⫾ 1938 (70% of the total dye uptake) after
cessation of the stimulus. This increase was four times greater
than the value with the 10 Hz stimulus (Figs. 4 B, 1C). Even when
the duration of the stimulus was reduced to 1 min, rather than 3
min, substantial dye uptake occurred, mostly after cessation of
the stimulus (data not shown). These results suggest that some
signal produced by 100 Hz stimulation, but not 10 Hz, triggers
this delayed endocytosis. The vesicles labeled during the delayed
endocytosis might become part of the reserve pool of vesicles,
which has been reported to be labeled by such intense stimulation
(Richards et al., 2000, 2003). Another interesting observation was
that, during the stimulation period, less dye was taken up at 100
Hz than at 10 Hz (Figs. 4 B, 1C), and the intense uptake only
occurred when the stimulus was stopped. This was true for different durations of stimulation (data not shown) suggesting that
some signal produced by 100 Hz stimulation suppresses this form
of endocytosis during the stimulus.
Recently, a brefeldin A (BFA)-sensitive, AP-3-mediated form
of endocytosis has been detected at mature mouse hippocampal
synapses (Scheuber et al., 2006; Voglmaier et al., 2006; Danglot
and Galli, 2007; Voglmaier and Edwards, 2007). A form of vesicle
cycling and FM1-43 dye uptake that is sensitive to BFA or L-type
calcium channel blockers has also been observed in immature
mouse and chick motor axons before myotube contact (Zakharenko
et al., 1999; Hata et al., 2007) (K. Hata and L. T. Landmesser,
unpublished observations). Thus, we further characterized this
delayed endocytosis by applying either BFA (10 ␮g/ml) or nifedipine (5 ␮M). Both BFA and nifedipine reduced the mean pixel
intensity of endplates to 12,868 ⫾ 656 (nend ⫽ 136; Nexp ⫽ 7)
( p ⬍ 0.05) and 10,009 ⫾ 739 (nend ⫽ 79; Nexp ⫽ 5) ( p ⬍ 0.01),
respectively (Fig. 4 B). We were also interested to determine
whether this delayed endocytosis was coupled to exocytosis.
Thus, BoNTXA, which cleaves the vesicle fusion protein SNAP25, and thus prevents undocked vesicles from docking with the
surface plasma membrane before exocytosis, was used. BoNTXA
1098 • J. Neurosci., January 19, 2011 • 31(3):1093–1105
Maeno-Hikichi et al. • Synaptic Vesicle Endocytosis/Exocytosis at Mouse NMJs
significantly reduced the poststimulus dye
uptake (mean pixel intensity after wash
was reduced to 11,285 ⫾ 1100) (nend ⫽ 50;
Nexp ⫽ 4) ( p ⬍ 0.01) (Fig. 4 B). Together,
our results suggest that different modes of
endocytosis occur at 10 and 100 Hz stimulation both during and after stimulation.
The difference in dye loss with 10 Hz
versus 100 Hz stimulation persists even
though a different pool of vesicles was
loaded with the 100 Hz stimulation
We then determined how this pool of vesicles loaded with 100 Hz stimulation was
mobilized by either 10 or 100 Hz stimulation. With either 10 or 100 Hz trains, little
dye was lost even after 4000 stimuli (Fig.
4C). This indicates that the vesicles loaded
with 100 Hz stimulation belong to a different pool than those loaded with 10 Hz
and that they are not immediately available for subsequent release. However, after 4000 stimuli, the 10 Hz stimulation
began to result in dye loss, whereas little
dye was lost with the 100 Hz stimulation,
even after 9000 stimuli (Fig. 4C). The
large error bars after 4000 stimuli at 10 Hz
were the result of different endplates starting to unload at different time points.
We next provided a 2 h rest period after the 100 Hz loading, as other studies
have shown that the pool of vesicles
Figure 4. The FM1-43 destaining with 10 or 100 Hz stimulation after loading at 100 Hz. A, FM1-43 uptake with a 100 Hz, 3 min
loaded with such intense stimulation, stimulation. B, A bar graph of mean pixel intensity after completion of uptake with or without 5 min poststimulus dye incubation.
probably as a result of bulk endocytosis, Mean pixel intensity values were 5151 ⫾ 343 (n ⫽ 91; N ⫽ 3) with uptake only during stimulation and 17,016 ⫾
end
exp
requires additional time to become com- 1595 (nend ⫽ 68; Nexp ⫽ 7) with an additional 5 min poststimulus uptake. BFA reduced the poststimulus uptake intensity to
petent to undergo exocytosis with stimu- 12,868 ⫾ 656 (nend ⫽ 136; Nexp ⫽ 7), nifedipine reduced it to 10,009 ⫾ 739 (nend ⫽ 79; Nexp ⫽ 5), and BoNTXA reduced it to
lation (Richards et al., 2000; Clayton et al., 11,285 ⫾ 1100 (nend ⫽ 50; Nexp ⫽ 4). Muscles were preincubated with drug before the start of uptake and kept in the drug during
2009). As shown in Figure 4 D, after such a and after stimulation. C, FM1-43 unloading with 10 or 100 Hz stimulus after 100 Hz loading. Black circles, 100 Hz (nend ⫽ 5; Nexp ⫽
2⫹
rest period, the labeled vesicles were now 5). White circles, 10 Hz (nend ⫽ 3; Nexp ⫽ 3). D, After 100 Hz loading, muscles were allowed to rest 2 h in low-Ca Tyrode’s
solution
after
which
they
were
subjected
to
either
10
or
100
Hz
stimulation.
After
this
rest
period,
vesicles
that
were
loaded with
able to be released, but once again the
100
Hz
stimulation
became
available
for
destaining,
but
considerably
less
dye
was
lost
at
100
Hz
compared
with
10
Hz.
Black circles
amount of destaining at 100 Hz was conand black triangles, 100 Hz (nend ⫽ 5; Nexp ⫽ 5). White circles and white triangles, 10 Hz (nend ⫽ 5; Nexp ⫽ 5). The black and white
siderably less than that at 10 Hz. In this
circles were from the same endplates, but the stimulation frequency was switched from 100 to 10 Hz at the 9000th stimulus. The
same series of experiments, the stimulus white and black triangles were from the same endings, but the stimulation frequency was switched from 10 to 100 Hz at the 9000th
frequency was switched from 10 to 100 Hz stimulus.
(Fig. 4 D, white and black triangles, respectively) or 100 to 10 Hz (Fig. 4 D, black
(2 mM), the EPP amplitude was not altered by folimycin (Fig. 5A).
and white circles, respectively) at the 9000th stimulus. In both
During a single 100 Hz train, EPP amplitudes declined to ⬃60%
cases, this resulted in an immediate switch in the destaining beof the starting value because of depression; however, this decline
havior (Fig. 4 D). These results indicate that, even though a difwas not significantly increased by folimycin (Fig. 5B). These obferent pool had presumably been loaded by the 100 Hz compared
servations suggest that either there is no rapid reuse of vesicles
with 10 Hz loading protocol, different amounts of dye were lost
during such short stimulations or that the proportion of vesicles
when these vesicles were unloaded with 10 Hz versus 100 Hz
reused is sufficiently small compared with those that are mobistimulation.
lized de novo so as to be undetectable. At vertebrate NMJs, the
estimated total number of vesicles is ⬃250,000 (Elmqvist and
Rapid reuse of vesicles is triggered by 100 Hz stimulation
Quastel,
1965; Schofield and Marshall, 1980; Reid et al., 1999),
We next attempted to assess whether rapid reuse of vesicles was
which
is
considerably larger than the vesicle number at many
responsible for the much smaller amount of dye loss with 100 Hz
central
synapses,
which may be on the order of several hundred.
stimulation by applying folimycin (Ertunc et al., 2007). FolimyThus, the contribution of this large vesicle pool might contribute
cin disrupts the proton gradient across the vesicle membrane and
more to maintaining effective neuromuscular transmission with
thus prevents the refilling with transmitter of previously used
high-frequency stimulation than the rapid reuse of vesicles. In
vesicles. When such vesicles undergo exocytosis, they do not refact, many studies have shown that the reserve pool is recruited
lease any transmitter, which results in a decline in EPP amplitude.
more effectively with intense stimulation at NMJs (Rizzoli and
During a single 1 s train at 10 Hz and at the normal level of Ca 2⫹
Maeno-Hikichi et al. • Synaptic Vesicle Endocytosis/Exocytosis at Mouse NMJs
J. Neurosci., January 19, 2011 • 31(3):1093–1105 • 1099
Betz, 2005). To determine whether sustained periods of stimulation resulted in
an increase in vesicle reuse or not, we
stimulated with 1 s trains interspersed
with 1 s rest periods for 10 min at 10 Hz
(3000 stimuli) or 1 min at 100 Hz (3000
stimuli) in the presence or absence of folimycin and measured the area under the
EPPs as well as any asynchronous release
during either the stimulation or resting
periods and calculated the quantal content as described earlier. We also corrected for the overestimation of summed
quantal content caused by the superimposition of EPPs at 100 Hz as described previously. Since folimycin did not affect the
increase in baseline because of the superimposition of EPPs, we reduced the value
at 100 Hz by 30% as we had done for the
data presented in Figure 2C. This corrected summed quantal content value was
normalized with the value obtained without folimycin at the 3000th stimuli for
both 10 and 100 Hz and plotted as shown
in Figure 5, C and D. Whereas there was
little effect of folimycin at 10 Hz (Fig. 5C),
at 100 Hz, there was a ⬃40% decline in the
summed quantal content in the presence
of folimycin (Fig. 5D). The summed
quantal content values at the 3000th stimulus with 100 Hz stimulation, were
33,219 ⫾ 5734 and 22,324 ⫾ 7457 without
and with folimycin, respectively, and were
significantly different ( p ⬍ 0.01). Even as
early as the 200th stimulus, the decline in
summed quantal content caused by folimycin was obvious (6993 ⫾ 1159 and
3889 ⫾ 967 without and with folimycin,
respectively; p ⬍ 0.01). These results suggest that stimulation at 100 Hz but not 10
Hz triggers a rapid reuse of vesicles that
had undergone either a kiss-and-run or a
very rapid full collapse followed by a rapid
endocytosis. Similar rapid recycling has
been observed in hippocampal synapses
(Harata et al., 2006a).
Next, we investigated the role of dynamin in transmission at 10 and 100 Hz
by applying the dynamin I and II GTPase
inhibitor, dynasore. Dynamin acts on the
process of synaptic vesicle scission from
the plasma membrane, which requires
4
Figure 5. The effect of folimycin and dynasore on EPP amplitude with 10 or 100 Hz stimulation. A, The ratio of the amplitude of
the nth/first EPP with a 10 Hz single train in the presence of (white circles; nend ⫽ 4; Nexp ⫽ 4) or absence of (black circles; nend ⫽
14; Nexp ⫽ 14) folimycin. B, Similar EPP amplitude ratio with a 100 Hz single train with (white circles; nend ⫽ 8; Nexp ⫽ 8) or
without (black circles; nend ⫽ 19; Nexp ⫽ 19) folimycin. C, Normalized summed quantal content for 10 Hz stimulation without
folimycin (white circles; nend ⫽ 4; Nexp ⫽ 4) and with folimycin (black circles; nend ⫽ 5; Nexp ⫽ 5). The summed quantal content
values were normalized to the value of the 3000th stimulus without folimycin. D, Normalized summed quantal content for 100 Hz
without folimycin (white triangles; nend ⫽ 5; Nexp ⫽ 5) and with folimycin (black triangles; nend ⫽ 5; Nexp ⫽ 5). The summed
quantal content values were normalized to the value of the 3000th stimulus without folimycin. The data for 10 Hz without
folimycin and 100 Hz without folimycin in C and D are the same data set as shown in Figure 2C but normalized to the value at the
3000th stimulus. E, An example of a train of EPPs with 100 Hz stimulation recorded from a control NMJ (top) and a different
dynasore-treated NMJ (bottom). The first 10 responses and
the last 10 responses are shown. Dynasore treatment inhibited
facilitation and greatly enhanced depression. F, The ratio of
the amplitude of the fifth/first EPP with either a 10 or 100 Hz
single train in the presence of (gray; nend ⫽ 7; Nexp ⫽ 2) or
absence of (black; nend ⫽ 10; Nexp ⫽ 2) dynasore. G, Similar
EPP amplitude ratio of last/first EPP with either a 10 or 100 Hz
single train with (gray; nend ⫽ 7; Nexp ⫽ 2) or without (black;
nend ⫽ 10; Nexp ⫽ 2) dynasore.
Maeno-Hikichi et al. • Synaptic Vesicle Endocytosis/Exocytosis at Mouse NMJs
1100 • J. Neurosci., January 19, 2011 • 31(3):1093–1105
GTP hydrolysis, and thus is an important
regulator of endocytosis (Xu et al., 2008;
Lu et al., 2009; Tsai et al., 2009; Chung et
al., 2010). Interestingly, dynasore strongly
enhanced depression at 100 Hz stimulation compared with control, even during
the initial stimulus trains. However, it had
little effect on EPP amplitude during 10
Hz stimulation (Fig. 5E–G). These data
suggest that, at 100 Hz, but not at 10 Hz,
dynamin-dependent endocytosis was required to maintain effective transmission.
However, even after prolonged stimulation, sufficient to deplete the pool of releasable vesicles, at both 10 and 100 Hz,
transmission was not completely abolished by dynasore, and at 10 Hz, gradual
recovery was seen (data not shown).
These data suggest that there may be a
dynamin-independent form of endocytosis in mouse NMJs. We cannot at present
exclude that another isoform of dynamin
that is not inhibited by dynasore, which
inhibits dynamin 1 and 2, mediates endocytosis. Alternatively, the sites on dynamin that are blocked by dynasore may
not always be accessible in the intact presynaptic terminal, and these might be more
accessible during the form of endocytosis
that is triggered by 100 Hz stimulation. The
existence of both dynamin-dependent and
-independent endocytosis has been reported in the calyx of Held (Xu et al., 2008),
in superior cervical ganglion neurons (Lu et
al., 2009), in bovine chromaffin cells (Tsai et
al., 2009), and the pool of vesicles responsible for spontaneous transmitter release was
recently reported to be dynamin independent in hippocampal GABAergic synapses
(Chung et al., 2010). A more detailed investigation of the roles of dynamin at 10
and 100 Hz stimulation is currently under
study. However, together, our data indicate that 100 Hz stimulation triggers the
rapid reuse of vesicles and that transmission at 100 Hz is much more sensitive to
dynamin inhibition than that at 10 Hz,
even for the same number of stimuli.
Figure 6. The MLCK pathway may act to trigger a switch in the mode of dye destaining used during 10 Hz versus 100 Hz
stimulation. A, Schematic model of suggested cellular and molecular mechanisms for dye loss at 10 and 100 Hz. A MLCK and myosin
II-regulated pathway is needed to maintain synaptic transmission at high-frequency stimulation but not at low-frequency stimulation. B, FM1-43 destaining at 10 Hz in the presence of a phorbol ester (red triangles; nend ⫽ 9; Nexp ⫽ 9) compared with the
control destaining with 100 Hz (filled circles) and 10 Hz (open circles). C, FM1-43 destaining at 10 Hz with 10 mM extracellular
calcium (red triangles; nend ⫽ 7; Nexp ⫽ 7) compared with destaining at either 100 or 10 Hz in 2 mM Ca 2⫹. D, FM1-43 destaining
at 10 Hz in the presence of a MLCK agonist peptide (red triangles; nend ⫽ 5; Nexp ⫽ 5) compared with control destaining at 100 or
10 Hz. The 100 Hz data and the 10 Hz data in B–D were the same as in Figure 2A and are shown here for comparison. E, Bar graph
of FM1-43 mean pixel intensity remaining in endplates after completion of 30 min of a 10 Hz destaining in the presence of the
phorbol ester PDBu, 10 mM calcium, or the MLCK agonist. The mean pixel intensities were 5820 ⫾ 386 (nend ⫽ 83; Nexp ⫽ 5) for
control, 12,735 ⫾ 1165 (nend ⫽ 42; Nexp ⫽ 5) for PDBu, 10,000 ⫾ 877 (nend ⫽ 65; Nexp ⫽ 5) for 10 mM calcium, and 7359 ⫾ 603
(nend ⫽ 75; Nexp ⫽ 5) for the MLCK agonist peptide. F, Normalized summed quantal content measured as in Figure 5, C and D.
White circles, 10 Hz without folimycin (n ⫽ 4; same as Fig. 5C for comparison); white triangles, 10 Hz with the MLCK agonist
peptide (nend ⫽ 4; Nexp ⫽ 4); black squares, 10 Hz with the MLCK agonist and folimycin (nend ⫽ 5; Nexp ⫽ 5).
A MLCK–myosin II pathway is required for the mode of
synaptic vesicle cycling that loses less dye and that is used
during 100 Hz stimulation
A MLCK signaling pathway (Fig. 6 A), shown to be required to
maintain effective transmission at high stimulation frequencies,
was initially discovered through studies of neural cell adhesion
molecule (NCAM)-deficient mouse NMJs, which were unable to
maintain effective transmission at high stimulus repetition rates
and also exhibited cyclical periods of total transmission failures
when stimulated at 100 Hz and above (Polo-Parada et al., 2001).
Subsequent experiments demonstrated that such total transmission failures at 100 Hz stimulation could be produced in wildtype NMJs by blocking MLCK with either ML-9 (Polo-Parada et
al., 2004) or by the introduction of a peptide inhibitor of MLCK
into the presynaptic terminal (Polo-Parada at al., 2005). Blocking
downstream targets of MLCK, for example MLC (the regulatory
light chain of myosin), with an inhibitor peptide or blocking
myosin II with the myosin II ATPase-specific inhibitor, blebbistatin (Polo-Parada et al., 2004, 2005), also caused transmission
failures with high-, but not low-frequency stimulation, further
supporting the importance of the MLCK–myosin II pathway in
maintaining synaptic transmission with high, but physiological
(Gorassini et al., 2000), stimulus frequencies.
The fact that the requirement for the MLCK pathway to prevent transmission failures was frequency dependent (PoloParada et al., 2005), together with the different modes of vesicle
cycling we observed with 10 or 100 Hz stimulation, led us to
hypothesize that the MLCK pathway might be needed to engage
the mode of vesicle recycling used at high frequency that loses less
Maeno-Hikichi et al. • Synaptic Vesicle Endocytosis/Exocytosis at Mouse NMJs
dye. A previous study (Polo-Parada et al., 2004) also provided
evidence that PKC activates the MLC–myosin II pathway possibly by inhibition of MLC-P (myosin light chain phosphatase). To
see whether activation of PKC could engage the MLCK pathway,
even during 10 Hz stimulation, and thus alter the mode of cycling, either of the phorbol esters, PMA or phorbol 12,13dibutyrate (PDBu), was applied to activate PKC during dye
destaining at 10 Hz stimulation. Normally, 10 Hz destaining
caused rapid dye loss (Fig. 2 A), and by the end of the stimulation,
the total mean pixel intensity was reduced to approximately onehalf (5820 ⫾ 386; nend ⫽ 83; Nexp ⫽ 5) of the fully loaded value
(Fig. 1C). However, phorbol ester treatment resulted in less dye
loss, usually seen only with 100 Hz stimulation. Significantly
( p ⬍ 0.01) more dye remained (mean pixel intensity, 12,735 ⫾
1165; nend ⫽ 42; Nexp ⫽ 5) after the 9000th stimulus (Fig. 6 B, E).
These observations suggest that, even with 10 Hz stimulation,
PKC activation is able to change the vesicle release mode to a
mode in which less dye is lost. In summary, activation of the
MLCK pathway alone, is sufficient to prevent transmission failures at 100 Hz and also to trigger a mode of vesicle cycling in
which less dye is lost.
Next, since the MLCK pathway is regulated by Ca 2⫹ (Fig. 6 A),
we speculated that an upstream signal that initiates the activation
of this pathway might be an increase in calcium concentration
within the presynaptic terminal. To test the idea, we increased the
calcium concentration in the bath from 2 to 10 mM and examined
the FM dye destaining with 10 Hz stimulation (Fig. 6C,E). This
resulted in much less dye being released than during the 10 Hz
destaining in 2 mM Ca 2⫹, with significantly ( p ⬍ 0.01) more dye
remaining after the 9000th stimulus (mean pixel intensity,
10,000 ⫾ 877; nend ⫽ 65; Nexp ⫽ 5) (Fig. 6 E). These data suggest
that a higher calcium concentration within the presynaptic terminal was able to prevent the fast dye loss mode of vesicle exocytosis even with 10 Hz stimulation and to shift the behavior of
release to one that loses less dye, which is usually only observed
with high-frequency stimulation.
Next, we tested the effect of activating MLCK by the MLCK
agonist peptide, which was previously shown to rescue the total
transmission failures in NCAM 180-deficient NMJs when stimulated at high frequency (Polo-Parada et al., 2005). This peptide
was also shown to cause phosphorylation of MLC in PC12 cells in
the absence of any stimulation, confirming that it caused MLCK
to be activated constitutively (Polo-Parada et al., 2005). Introducing the MLCK agonist peptide with the peptide carrier Chariot resulted in less dye loss even at 10 Hz stimulation (Fig. 6 D)
and considerably more dye remained after 9000 stimuli (mean
pixel intensity, 7359 ⫾ 603; nend ⫽ 75; Nexp ⫽ 5) (Fig. 6 E), which
differed significantly from the normal 10 Hz destaining ( p ⬍
0.05). Our electrophysiological recordings indicated that quantal
content was similar when NMJs were treated with the MLCK
agonist (42.53 ⫾ 5.68 and 42.16 ⫾ 5.19, with or without the
MLCK agonist, respectively), suggesting again that transmitter
release was normal but FM1-43 dye release was less, similar to
that occurring normally with 100 Hz stimulation.
We then tested whether the greater retention of dye that occurred with MLCK activation at 10 Hz was caused by the rapid
reuse of vesicles, which we had observed by using folimycin during 100 Hz stimulation. Thus, we activated MLCK and then stimulated NMJs at 10 Hz, with or without folimycin, and measured
the summed quantal content. Activation of MLCK alone did not
change the summed quantal content (Fig. 6 F, white circle and
triangle); however, MLCK activation together with folimycin
strongly reduced the value of the summed quantal content. This
J. Neurosci., January 19, 2011 • 31(3):1093–1105 • 1101
Figure 7. Phospho-MLC is upregulated with PMA treatment or with a 100 Hz stimulus. A,
Immunostaining of adult NMJs. Endplates were stained with ␣-bungarotoxin (␣-BTX), a
postsynaptic marker, and a phosphospecific myosin light chain (p-MLC) antibody. The p-MLC
staining for PMA treated endings and those stimulated at 100 Hz were upregulated compared
with unstimulated controls or those stimulated at 10 Hz. Scale bar, 10 ␮m. B, Cumulative
distribution of p-MLC staining with various treatments. PMA and 100 Hz stimulus increased the
p-MLC staining greatly. The mean values are as follows: control, 1142 ⫾ 168 (nend ⫽ 45; Nexp ⫽ 3);
PMA, 3493 ⫾ 499 (nend ⫽ 49; Nexp ⫽ 4); 10 Hz, 1443 ⫾ 208 (nend ⫽ 48; Nexp ⫽ 3); 100 Hz,
2904 ⫾ 399 (nend ⫽ 53; Nexp ⫽ 4). The statistical significance determined by the Kolmogorov–
Smirnov test compared with the unstimulated control was p ⬍ 0.01 for 10 Hz, p ⬍ 0.001 for
100 Hz, and p ⬍ 0.001 for PMA. The values for PMA and 100 Hz stimulation did not differ
significantly, p ⫽ 0.765.
observation demonstrates more directly that activation of the
MLCK pathway increases the number of vesicles that are rapidly
recycled and reused.
High-frequency stimulation or PMA increases the
phosphorylation of myosin II light chain in intact endplates
Activating the MLCK pathway with the MLCK agonist peptide
altered the amount of dye loss as well as the mode of vesicle
recycling. To see whether changes in the activation of the MLCK
pathway actually occurred in intact endplates when their axons
were stimulated with 10 or 100 Hz stimuli or with phorbol ester,
immunostaining with an antibody that recognizes MLC when it
is phosphorylated on serine 19 and threonine 18 was performed.
Phosphorylation by MLCK at these sites on MLC results in activation of myosin II (Somlyo and Somlyo, 2003). Thus, phosphoMLC (p-MLC) staining can be used as an indicator of the state of
activation of the MLCK pathway in intact nerve terminals. Im-
1102 • J. Neurosci., January 19, 2011 • 31(3):1093–1105
Maeno-Hikichi et al. • Synaptic Vesicle Endocytosis/Exocytosis at Mouse NMJs
munostaining for p-MLC was performed
on frozen sections from semitendinosus
muscles that had previously been placed
in oxygenated Tyrode’s solution and either not treated, treated with PMA, or
stimulated at different frequencies. In
nonstimulated muscles, p-MLC staining
was present (Fig. 7A) as previously reported (Polo-Parada et al., 2005), but as
evident from the distribution of endplates
with different mean pixel intensities, most
had low levels of p-MLC (Fig. 7B). When
muscles were treated with 100 nM PMA
and rapidly fixed in the presence of phosphatase inhibitors, staining was more intense (Fig. 7A) and the distribution of
mean pixel intensities was shifted to
higher values (Fig. 7B). This result confirmed that PKC activation led to MLCK
activation at NMJs. Next, muscles were either stimulated at 10 or 100 Hz for several
minutes and rapidly fixed toward the end
of the stimulation. Stimulation at 10 Hz
increased p-MLC staining only slightly
above control levels (Fig. 7 A, B). However, stimulation at 100 Hz resulted in a
clear shift in the distribution of p-MLC
levels to higher values (Fig. 7 A, B), similar
to that caused by PMA. Thus, these observations show that 100 Hz, but not 10 Hz,
stimulation strongly activates the MLCK
signaling pathway and could serve as the
signal to switch to the myosin IIdependent form of vesicle cycling that is
engaged at high stimulus frequencies.
PKC␧ regulates the MLCK pathway
Among the PKC isoforms, PKC␧ is more
concentrated in the nervous system (Ono
et al., 1988; Koide et al., 1992; Wetsel et al., Figure 8. PKC␧ regulates the activation of the MLCK pathway and the effectiveness of synaptic transmission. A, Endplates were
1992) and is unique in containing an actin stained with ␣-BTX (a postsynaptic marker) and with a PKC␧ antibody. The top panel shows an entire endplate from a 50 ␮m
binding domain (Prekeris et al., 1996). It longitudinal section viewed from above. The bottom panel is taken from a 12 ␮m transverse section of the muscle and shows the
is known to be involved in voltage-gated gutters of several endplates visualized with ␣-BTX. The PKC␧ immunostaining is located more peripherally in a presynaptic
calcium channel signaling (Maeno-Hikichi et location. Scale bar, 10 ␮m. B, Phospho-MLC staining at 10 or 100 Hz stimulated endings that were pretreated with a PKC␧
al., 2003), inducing synaptic potentiation translocation inhibitor peptide. Staining was greatly reduced with 100 Hz stimulation after the PKC␧ inhibitor peptide treatment
and was similar to the control value at 10 Hz. Scale bar, 10 ␮m. C, Cumulative distribution of p-MLC staining at 10 or 100 Hz
at the calyx of Held (Saitoh et al., 2001), stimulus pretreated with PKC␧ inhibitor peptide. The mean values are as follows: 10 Hz, 1072 ⫾ 145 (n ⫽ 52; N ⫽ 3); 100
end
exp
regulating glutamate exocytosis from syn- Hz, 1657 ⫾ 216 (n ⫽ 46; N ⫽ 3). Statistical significance was determined by Kolmogorov–Smirnov
test and was not
end
exp
aptosomes (Prekeris et al., 1996) and the significant, p ⫽ 0.06. D, A summary bar graph of mean pixel intensities for p-MLC immunostaining of endings under different
secretion of neuropeptides from Caeno- experimental conditions. PMA treatment and 100 Hz stimulation increased the p-MLC level, but pretreatment with the PKC␧
rhabditis elegans motor neurons (Sieburth inhibitor peptide prevented this increase. E, Traces of EPPs during a 1 s 100 Hz train in control or PKC␧ inhibitor peptide-pretreated
NMJs. Some endings (middle trace) exhibited depression and greater variability in EPP amplitude than the controls (top trace).
et al., 2007).
To see whether PKC␧ is present in Others exhibited cyclical total transmission failures, in this case to every other stimulus (bottom trace).
adult NMJs, immunostaining of adult
compared with 10 Hz stimulation without the inhibitor peptide
mouse junctions was performed using a PKC␧-specific antibody.
(Fig. 7A). However, the inhibitor treatment reduced the intensity
PKC␧ was localized in a punctate manner at endplates, which were
of p-MLC staining with 100 Hz stimulation to that seen with 10
visualized with ␣-bungarotoxin staining for postsynaptic acetylchoHz stimulation. (Fig. 8 B, C). These observations show that, durline receptors (Fig. 8 A). Staining of cross-sections showed that
ing 100 Hz stimulation, PKC␧ is important in causing the phosPKC␧ was abundant in the presynaptic terminal (Fig. 8 A). To test
phorylation of MLC. These results are summarized in the bar
whether PKC␧ played a role in MLC phosphorylation with 100
graph of Figure 8 D. It is clear that both PMA and 100 Hz stimuHz stimulation, NMJs were pretreated with a PKC␧ translocation
lation increase the phosphorylation level of MLC and that preinhibitor, and then stimulated at either 10 or 100 Hz, fixed, and
treatment with the PKC␧ inhibitor inhibited the increased
then immunostained with the p-MLC antibody. With 10 Hz
stimulation, there was no effect of the PKC␧ inhibitor (Fig. 8 B, C)
phosphorylation in response to the 100 Hz stimulus. To deter-
Maeno-Hikichi et al. • Synaptic Vesicle Endocytosis/Exocytosis at Mouse NMJs
mine whether the PKC␧ inhibitor affected vesicle release, electrophysiological recordings were made (Fig. 8 E). Interestingly, we
found a heterogeneous response between endings when stimulated at 100 Hz. One group (Fig. 8 E, bottom) exhibited cyclical
total transmission failures, in this example failing with every
other stimulus, whereas the other (Fig. 8 E, middle) exhibited
depression and greater variation in EPP amplitude than in control 100 Hz trains but no failures.
J. Neurosci., January 19, 2011 • 31(3):1093–1105 • 1103
We showed here that at adult mouse NMJs different modes of
exocytosis and endocytosis are used for transmitter release, depending on the frequency of stimulation. At 10 Hz, the speed of
vesicle exocytosis and endocytosis appear balanced and the behavior of both transmitter and FM dye release were similar, suggesting full collapse exocytosis and subsequent endocytosis for
vesicle replenishment. In contrast, at 100 Hz, although transmitter continued to be released at sufficient levels to maintain effective transmission, less FM dye was released than predicted by the
summed quantal content. This lower amount of dye loss suggests
that the actual release mechanism may have changed with highfrequency stimulation. Applying folimycin during the 100 Hz
stimulation resulted in much less transmitter being released than
without the drug, indicating that, at 100 Hz, vesicles are recycled
and reused much more rapidly than at 10 Hz. Also, the dynamin
inhibitor, dynasore, greatly enhanced depression at 100 Hz with
much less effect at 10 Hz. Thus, dynamin-sensitive endocytosis
contributed much more to maintaining effective transmission
with 100 Hz than with 10 Hz stimulation. However, dynamin did
not block transmission completely.
Blocking the MLCK pathway during high-frequency stimulation was previously shown to cause total transmission failures,
presumably because of an inability to exocytose release competent vesicles at a rate needed to maintain transmission (PoloParada et al., 2005). Here, we provide direct evidence that
stimulation at 100 Hz, but not 10 Hz, activates the MLCK pathway, as evidenced by increased phospho-MLC immunostaining
in the nerve terminal. These observations lead us to conclude that
a synaptic activity-dependent activation of MLCK is a key regulator for triggering a switch in the mode of vesicle cycling to
facilitate recycling of vesicles during high-frequency stimulation.
We also provide evidence that an elevated presynaptic calcium
concentration and an upregulation of PKC activity, especially of
PKC␧, enhanced MLCK activation. The treatments that led to
MLCK activation also caused a switch to the mode of vesicle
exocytosis that lost less dye, even when junctions were stimulated
at 10 Hz. In addition, inhibition of PKC␧ caused total transmission failures at 100 Hz, similar to those previously reported in 180
NCAM-deficient mice, which exhibit impaired MLCK activation
(Polo-Parada et al., 2005).
(Zhang et al., 2009; Zhu et al., 2009), will be needed to definitively
determine the type of vesicle fusion used during the rapid reuse
we have observed.
In addition, we clearly showed that, even with 10 Hz stimulation, activation of the MLCK–myosin II pathway caused both a
switch to a mode of exocytosis that lost less dye and an increase in
the proportion of vesicles that were rapidly recycled. An enzymatic balance between MLCK and myosin light chain phosphatase regulates myosin II activity through phosphorylation of its
light chain. MLCK in turn is activated by calcium and calmodulin
(CaM), whereas myosin light chain phosphatase is inhibited by a
peptide, CPI-17, which is activated by PKC (Somlyo and Somlyo,
2003) (Fig. 6 A). Thus, during high-frequency stimulation, it is
likely that calcium influx would activate both the CaM and PKC
signaling pathways, leading to the activation of MLCK and the
inhibition of myosin phosphatase, respectively. The 180 isoform
of NCAM, acting via a conserved intracellular domain, is also
required for activation of the MLCK pathway by high-frequency
stimulation (Polo-Parada et al., 2005). However, how NCAM
enables activation of this pathway remains to be determined.
Myosin II has also been shown to regulate catecholamine and
neuropeptide release (Chan et al., 2005; Doreian et al., 2008,
2009; Berberian et al., 2009) as well as vesicle transport and fusion
pore expansion in chromaffin cells (Neco et al., 2004, 2008). At
the calyx of Held synapse, inhibition of MLCK was reported to
increase the size of the fast releasing vesicle pool (Srinivasan et al.,
2008). How the MLCK–myosin II pathway regulates the release
of synaptic vesicles appears to vary among the systems studied,
suggesting multiple roles of this pathway in different systems.
Although we have clearly demonstrated that the MLCK–myosin II pathway is required to maintain transmission with highfrequency stimulation, and that it enables the rapid reuse of
vesicles and a form of exocytosis in which less FM dye is lost,
determining the exact roles of myosin II in these processes will
require additional studies. In addition to its role as a motor protein, myosin II has been shown to promote the severing of actin
filaments and thus influence actin treadmilling in growth cones,
as well as actin cytoskeleton dynamics in a variety of cell types
(Lin et al., 1996; Medeiros et al., 2006; Vallee et al., 2009; VicenteManzanares et al., 2009; Rex et al., 2010). Dynasore can also cause
remodeling of actin filaments by altering the association of dynamin and other molecules with the filaments (Mooren et al.,
2009; Yamada et al., 2009). Thus, the effect of dynasore we observed might be attributable to inhibition of endocytosis and/or
actin destabilization. Inhibition of actin polymerization can also
slow catecholamine release from chromaffin cells (Doreian et al.,
2008, 2009; Berberian et al., 2009). Clearly, much additional work
will be required to show how myosin II activity and the regulation
of actin dynamics affect exocytosis and the recycling of synaptic
vesicles.
Characteristics of the mode of vesicle cycling in which less dye
is lost
Our folimycin data support the idea of rapid reuse of vesicles with
high-, but not low-frequency stimulation. Such rapid reuse could
be achieved by a kiss-and-run/transient fusion pore form of exocytosis (but see Wu and Wu, 2009). Alternatively vesicles might
undergo a rapid full collapse and rapid retrieval and be returned
to the readily releasable pool. In either case, more FM dye would
be retained in the terminal than predicted by the number of
exocytosed vesicles based on the summed quantal content. Additional experiments, such as after the exocytosis of single vesicles
labeled with nanoparticles or with a fluorescent pH reporter
Why are total transmission failures observed when the
MLCK–myosin II pathway is inhibited during high-frequency
stimulation?
Adult mouse NMJs that lack all NCAM isoforms (Polo-Parada et
al., 2001) or only the 180 isoform (Polo-Parada et al., 2004, 2005)
exhibit total transmission failures when stimulated at 100 Hz or
above. These were shown to be attributable to events downstream
of Ca 2⫹ influx into the terminal (Polo-Parada et al., 2001, 2004)
and were prevented by activation of the MLCK pathway. In the
present study, such failures were caused by inhibiting PKC␧, presumably upstream of the MLCK pathway. We currently do not
Discussion
1104 • J. Neurosci., January 19, 2011 • 31(3):1093–1105
have a clear explanation for the transmission failures. However,
several observation made in the present study appear relevant.
One plausible hypothesis is that the failures are caused by
blockage of active zone release sites by the exocytosed vesicles,
which must be cleared to make room for subsequent vesicle docking. If exocytosis, and subsequent endocytosis, occur at different
places, the blockage of endocytosis with dynasore should have
only a gradual effect on exocytosis as recently endocytosed vesicles are reused. Thus, we expected to see a decline in EPP amplitude with a time course similar to that observed with folimycin
treatment. However, dynasore caused an immediate, large enhancement of depression with 100 Hz stimulation, but very little
with 10 Hz stimulation, suggesting that some of the recently exocytosed vesicles might be retrieved in a dynamin-dependent
manner, freeing release sites for subsequent exocytosis. Ca 2⫹/
calmodulin-initiated endocytosis was recently shown to facilitate
vesicle mobilization by clearing fused vesicle membrane at release
sites (Wu et al., 2009). Depression, caused by perturbation of
calcium-dependent coupling of exocytosis and endocytosis, has
also been suggested to result from a temporary reduction in new
vesicles docking at release sites (Hosoi et al., 2009). These reports
suggest that the physical availability of release sites might affect
both exocytosis and endocytosis.
Another challenge of explaining the transmission failures is
how the hundreds of active zones at rodent NMJs (Nishimune et
al., 2004) could all fail simultaneously. At rat and frog NMJs, the
probability of release for individual vesicles at each active zone is
low (Wachman et al., 2004; Rowley et al., 2007) and many active
zones contribute to the EPP. However, it was recently shown for
mouse NMJs that, during high- but not low-frequency stimulation, preferred sites of exocytosis exist and that these are very
close to sites of endocytosis (Gaffield et al., 2009a,b). If the probability of release at individual active zones changes during highfrequency stimulation and exocytosis only occurs at a limited
number of preferred sites, temporary blockage of those sites
would be more likely to produce total transmission failures. We
did not observe failures with dynasore treatment. However, the
persistence of a dynasore-insensitive form of transmission makes
interpretation of this result difficult. Additional experiments will
be needed to elucidate the mechanism underlying transmission
failures.
In conclusion, this study shows that activation of the MLCK–
myosin II pathway by high-frequency stimulation acts as a switch
to trigger alterations in exocytosis and endocytosis, which are
needed to maintain effective transmission. These include the
rapid reuse of vesicles and an altered form of exocytosis that loses
less FM dye. A full understanding of the precise mechanisms
responsible for this altered dye loss will be explored in future
experiments.
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